Screening methods using canine t2r receptors and pet food products and compositions identified using the same

ABSTRACT

The presently disclosed subject matter relates to methods of screening raw materials and pet food products to manufacture a palatable pet food product. The presently disclosed subject matter also relates to methods for identifying compounds that modulate the activity and/or expression of a bitter taste receptor.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. patent application Ser. No.15/746,658, filed on Jan. 22, 2018, which claims priority toInternational Patent Application No. PCT/US2016/044540, filed on Jul.28, 2016, which claims priority to U.S. Provisional Application Ser. No.62/197,983, filed on Jul. 28, 2015, the content of each are incorporatedby reference in their entireties, and to which priority is claimed.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 27, 2020, isnamed 0692690239CONSEQ.txt and is 65,750 bytes in size.

FIELD

The presently disclosed subject matter relates to the use of canine T2Rbitter taste receptors (cT2Rs) for the identification of T2R modulators.The presently disclosed subject matter further relates to the use ofcanine T2R bitter taste receptors to screen raw materials for making petfood products, as well as screening finished pet food products, for thepresence of T2R modulating compounds.

BACKGROUND

Taste profiles for edible compositions include basic tastes such assweet, salt, bitter, sour, umami and kokumi. Taste profiles have alsobeen described as including free fatty acid tastes. Chemical compoundsthat elicit these tastes are often referred to as tastants. Withoutbeing bound by theory, it is hypothesized that tastants are sensed bytaste receptors in the mouth and throat which transmit signals to thebrain where the tastants and resulting taste profiles are registered.Taste receptors include the T2R family of receptors, which comprise aG-protein coupled receptors (GPCR) family that detects compoundsassociated with bitter taste sensory perception.

Pet food manufacturers have a long-standing desire to provide pet foodproducts that have high nutritional value. In addition, and withparticular regard to cat and dog foods, pet food manufacturers desire ahigh degree of palatability so that pets can receive the fullnutritional benefit from their food. Domestic animals are notoriouslyfinicky in their food preferences, and often refuse to eat a pet foodproduct that it has accepted over time or refuse to eat any more than aminimal amount of a pet food product. This phenomenon may be, in part,due to the subtle differences in the sensory profiles of the rawmaterial, which can be perceived by the domestic animals because oftheir gustatory and olfactory systems. As a result, pet ownersfrequently change types and brands of pet food in order to maintaintheir pets in a healthy and contented condition.

While there have been recent advances in taste and flavor technologies,there remains a need for methods of screening raw materials that areused to make pet food product, and for screening finished pet foodproducts, to ensure that the most palatable products and processes formaking the pet food products are used. There also remains a need forcompounds that can enhance or modify the palatability of pet foodproducts by enhancing or modifying the taste, texture and/or flavorprofiles of the pet food products. The enhancement or modification canbe used to increase the intensity of a desirable attribute, to replace adesirable attribute that is not present or somehow lost in the pet foodproduct, or to decrease the intensity of an undesirable attribute. Inparticular, it is desirable to decrease the presence or intensity of anundesirable bitter tastant in a pet food product. Similarly, there is aneed to increase the acceptance of pet medications by enhancing ormodifying the palatability of the medications.

The pet care industry is also concerned with developing taste deterrentsthat can effectively discourage a pet from chewing, licking, oringesting things that are harmful to the health of the animal. While itis known that bitter taste can be effective to deter pets, there is asignificant variation in pets' reactions to these bitter tastedeterrents. Thus, there exists a need for compounds that effectivelyimpart an undesirable bitter taste to harmful or toxic objects.

Therefore, there remains a need in the art for methods to screen raw petfood materials (e.g. new protein sources), as well as final pet foodproducts, to provide palatable and nutritious pet food. There alsoremains a need to identify compounds that enhance, decrease, orotherwise modulate the palatability and/or bitter taste of pet foodproducts, or objects, and for flavor compositions comprising thesecompounds.

SUMMARY OF THE INVENTION

The presently disclosed subject matter provides methods for identifyingcompounds that enhance, increase, decrease and/or modulate the activityand/or expression of a bitter taste receptor. In certain embodiments,the methods entail screening for compounds that modulate the bitterreceptor activity and/or expression in a pet food product or medicine,or in raw materials used to make the pet food product or medicine. Thepresently disclosed subject matter also provides compounds that enhance,increase, decrease and/or modulate the activity and/or expression of abitter taste receptor identified by said methods. In certainembodiments, the bitter taste receptor is a T2R receptor. In otherembodiments, the bitter taste receptor is a canine T2R receptor.

In certain embodiments, the method for identifying compounds thatenhance, increase, decrease and/or modulate the activity and/orexpression of a bitter taste receptor comprises expressing a bittertaste receptor having a nucleotide sequence set forth in any one or moreof SEQ ID NOs: 1-16, or a fragment or variant thereof, in a cell. Themethod can further comprise contacting the cell expressing the bittertaste receptor with a sample (e.g., pet food raw material, finished petfood, or a test compound) and determining the activity and/or expressionof the bitter taste receptor in the presence of the sample as comparedto the activity and/or expression of the receptor in the absence of thesample. In certain embodiments, the activity and/or expression of thebitter receptor is determined in the presence of the sample and a bitterreceptor agonist.

In certain embodiments, a method for identifying compounds that enhance,increase, decrease and/or modulate the activity and/or expression of abitter taste receptor comprises expressing a bitter taste receptorhaving an amino acid sequence set forth in any one or more of SEQ IDNOs: 17-32, or a fragment or variant thereof, in a cell. The method canfurther comprise contacting the cell expressing the bitter tastereceptor with a sample (e.g., pet food raw material, finished pet food,or a test compound) and determining the activity and/or expression ofthe bitter taste receptor in the presence of the sample as compared tothe activity and/or expression of the receptor in the absence of thesample. In certain embodiments, the activity and/or expression of thebitter receptor is determined in the presence of the sample and a bitterreceptor agonist.

In certain embodiments, the present disclosure provides a method foridentifying a composition that modulates the activity of a bitter tastereceptor comprising (a) contacting a bitter taste receptor agonist witha bitter taste receptor, (b) determining the activity of the bittertaste receptor, (c) contacting a test agent with the bitter tastereceptor, (d) determining the activity of the bitter taste receptor, and(e) selecting the test agent as the composition when the activity of (d)is greater than or less than the activity of (b).

In certain non-limiting embodiments, the methods for identifying acompound that modulates the activity of a bitter taste receptordescribed herein utilize cells expressing a bitter receptor that isnative to the cells. Examples of such cells expressing a native bitterreceptor include, for example but not limited to, dog and/or cat tastecells (e.g., primary taste receptor cells). In certain embodiments, thedog and/or cat taste cells expressing a bitter receptor are isolatedfrom a dog and/or cat and cultured in vitro. In certain embodiments, thetaste receptor cells can be immortalized, for example, such that thecells isolated from a dog and/or cat can be propagated in culture.

The present disclosure also provides for methods for identifyingcompounds that enhance, increase, decrease and/or modulate the activityand/or expression of a bitter taste receptor, wherein the assay isconducted using a cell-free assay, for example, wherein the bitter tastereceptor is bound to or otherwise attached to a substrate.

The present disclosure also provides for methods for identifyingcompounds that enhance, increase, decrease and/or modulate the activityand/or expression of a bitter taste receptor, wherein the assay isconducted using an in silico model of the bitter taste receptor, forexample, wherein the bitter taste receptor is modeled using a computerprogram and binding of the compound to the receptor is predicted throughdocking algorithms.

The presently disclosed subject matter further provides a method formaking a palatable pet food product, wherein the raw materials used togenerate the pet food product are screened to determine if they containcompounds that enhance, increase, decrease and/or modulate the activityand/or expression of a bitter taste receptor. In certain embodiments,the raw material is a novel protein source. In certain embodiments theraw material is a protein source that is not commonly consumed in thehuman food chain. In certain embodiments, a raw pet food product thatcomprises a compound that increases the activity and/or expression of abitter taste receptor (for example, as compared to a bitter tastereceptor not contacted with the raw material) is not selected for use ingenerating a finished pet food product. In other embodiments, a raw petfood material that does not increase the activity and/or expression of abitter taste receptor (or that reduces the activity of a bitter tastereceptor, for example, in the presence of a bitter receptor agonist) isselected for generating a finished pet food product.

The presently disclosed subject matter further provides a method formaking a palatable pet food product, wherein the finished pet foodproduct is screened to determine if it contains compounds that enhance,increase, decrease and/or modulate the activity and/or expression of abitter taste receptor. In certain embodiments, the compounds are formedduring the manufacturing process. In one embodiment, a finished pet foodproduct that comprises a compound that increases the activity and/orexpression of a bitter taste receptor (for example, as compared to abitter taste receptor not contacted with the finished pet food product)is supplemented with one or more compounds that decrease the activityand/or expression of a bitter taste receptor (for example, an antagonistcompound).

The presently disclosed subject matter further provides a method formaking a palatable pet medicine product, wherein the finished petmedicine product is screened to determine if it contains compounds thatenhance, increase, decrease and/or modulate the activity and/orexpression of a bitter taste receptor. In certain embodiments, thecompounds are formed during the manufacturing process. In oneembodiment, a finished pet medicine product that comprises a compoundthat increases the activity and/or expression of a bitter taste receptor(for example, as compared to a bitter taste receptor not contacted withthe finished pet medicine product) is supplemented with one or morecompounds that decrease the activity and/or expression of a bitter tastereceptor (for example, an antagonist compound).

The presently disclosed subject matter further provides flavorcompositions that comprise a modulator of a bitter taste receptor, e.g.,an agonist and/or an antagonist and/or an allosteric modulator and/or aninverse agonist, identified according to the methods described herein.

In certain embodiments, said compounds can be used in methods formaintaining the health of an animal by imparting a bitter taste and/ordecreasing the palatability of an object or surface. In certainembodiments, the method comprises applying a taste deterrent productcomprising a compound as described herein to the object or surface. Incertain embodiments, the object is harmful to the health of the animalor toxic to the animal.

The foregoing has outlined rather broadly the features and technicaladvantages of the present application in order that the detaileddescription that follows may be better understood. Additional featuresand advantages of the application will be described hereinafter whichform the subject of the claims of the application. It should beappreciated by those skilled in the art that the conception and specificembodiment disclosed may be readily utilized as a basis for modifying ordesigning other structures for carrying out the same purposes of thepresent application. It should also be realized by those skilled in theart that such equivalent constructions do not depart from the spirit andscope of the application as set forth in the appended claims. The novelfeatures which are believed to be characteristic of the application,both as to its organization and method of operation, together withfurther objects and advantages will be better understood from thefollowing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows canine bitter taste receptor (T2R) nucleotide sequences(SEQ ID NOs: 1-16) along with their corresponding amino acid sequences(SEQ ID NOs: 17-32). The sequences include the canine bitter tastereceptors cT2R1, cT2R2, cT2R3, cT2R4, cT2R5, cT2R7, cT2R10, cT2R12,cT2R38, cT2R39, cT2R40, cT2R41, cT2R42, cT3R43, cT2R62, and cT2R67.

FIGS. 2A-2C show canine T2R sequence alignments. The dashed grey arrowsindicate active site positions occupied by mostly asparagine or serineresidues. The solid black arrows indicate structural tryptophan residuesthat are present in all human and cat bitter receptors as well as allcanine bitter receptors except T2R12. The dashed black arrows indicatethe conserved asparagine which is present in most of the bitterreceptors. The conjoined solid arrows indicate the conserved LxxxR motif(IxxxR for some instances in T2R2), wherein x can be any amino acid. Theconjoined dashed arrows indicate the conserved LxxSL motif.

FIG. 3A-E shows (A) the chemical structure of Menthol, (B) in silicomodeling of Menthol docked within the active site of the canine T2R1,(C) a close-up view of selected residues lining the active site pocketinteracting with, or close to, Menthol, (D) a ligand interaction mapdemonstrating potential interaction sites between Menthol and T2R1 and(E) a dose-response curve for Menthol when tested against canine T2R1 invitro. Asn89 can potentially make a hydrogen bond interaction with theligand. Other residues that can potentially make hydrogen bondinginteractions, pi interactions, or charged interactions with the ligandinclude Tyr239. Residues that can potentially make van der Waalsinteractions with the ligand include Ile167, Gln174, Glu169, Phe257,Ala242, Phe177, His238, Cys260, Phe264, Leu234, Cys235, Phe85, Leu261,Leu178, Leu181, Val86, and Phe82.

The backbone of the protein is represented as a ribbon to depict thehelical nature of the seven transmembrane-helix structure of thereceptor. The ligand is shown in space-filling CPK format (Corey et al.,Rev Sci Instrum, 24(8): 621-627 (1953)). In this and later FIGS. 3-9)hydrogen bond interactions with the ligand are shown in dotted lines,while salt-bridge and other interactions are shown as solid lines. Forthe interaction maps hydrogen bonding and other specific interactionsare shown as arrows, while residues forming a contact with the ligandare represented as circles. Darker circles represent residues with vander Waals interactions with the ligand, while lighter circles representresidues with polar, hydrogen bonding, Pi interactions, or chargedinteractions with the ligand. A lighter outer circle around a residue,if present, signals a large change in its solvent accessible surfacewhen the ligand binds. More residues are shown in the schematicinteraction maps in (D) than in the 3D model views in (C), sinceincluding all of the residues in (C) would obscure the view of theligand.

During ligand binding and receptor activation, active siterearrangements occur. As such, modeled interactions are dynamic, and maybe formed or break dynamically, and may be replaced with otherinteractions in the vicinity of the ligand during these processes.

FIG. 4A-E shows (A) the chemical structure of Ofloxacin, (B) in silicomodeling of Ofloxacin docked within the active site of the canine T2R2,(C) a close-up view of selected residues lining the active site pocketinteracting with, or close to, Ofloxacin, (D) a ligand interaction mapdemonstrating potential interaction sites between Ofloxacin and T2R2 and(E) a dose-response curve for Ofloxacin when tested against canine T2R2in vitro. Residues that can potentially make hydrogen bond or saltbridge interactions with the ligand include Ser94, Trp90, Lys268,Tyr245, and Glu180. Additional residues that can potentially make polar,hydrogen bonding, pi interactions, or charged interactions with theligand include Arg176 and Met91. Additional residues that canpotentially make van der Waals interactions with the ligand includeAsn185, Va1184, Met181, Phe249, Pro155, Gln177, Lys174, Phe264, Phe93,Leu59, Met271, Phe246, and Leu188.

FIG. 5A-E shows (A) the chemical structure of Chloroquine, (B) in silicomodeling of Chloroquine docked within the active site of the canineT2R3, (C) a close-up view of selected residues lining the active sitepocket interacting with, or close to, Chloroquine, (D) a ligandinteraction map demonstrating potential interaction sites betweenChloroquine and T2R3 and (E) a dose-response curve for Chloroquine whentested against canine T2R3 in vitro. Residues that can make hydrogenbonding or charged interactions with the ligand include Asn93 and Asp86.Additional residues that can make polar, hydrogen bonding, piinteractions, or charged interactions with the ligand include Tyr246,Phe247, Thr186, Asn189, Trp89, and Arg175. Additional residues makingprimarily van der Waals interactions with the ligand include Phe250,Glyl85, Phe243, Thr90, Asn176, Val149, Ile154, Lys174, Met82, Ile85,Lys173, and Met69.

FIG. 6A-E shows (A) the chemical structure of Colchicine, (B) in silicomodeling of Colchicine docked within the active site of the canine T2R4,(C) a close-up view of selected residues lining the active site pocketinteracting with, or close to, Colchicine, (D) a ligand interaction mapdemonstrating potential interaction sites between Colchicine and T2R4and (E) a dose-response curve for Colchicine when tested against canineT2R4 in vitro. Ser186, Asp93, and Tyr240 can potentially make a hydrogenbond with the ligand. Additional residues that can potentially makepolar, hydrogen bonding, pi interactions, or charged interactions withthe ligand include Ser94, Leu97, Asn95, Leu92, Ser96, Trp98, Val187, andThr247. Residues that can potentially make van der Waals interactionswith the ligand include Tyr243, Trp89, Met58, Ser269, Pro273, Ser270,Gln189, Thr144, Leu188, Val183, Leu182, Ser244, and Met90.

FIG. 7A-E shows (A) the chemical structure of 1, 10 Phenanthroline, (B)in silico modeling of 1,10 Phenanthroline docked within the active siteof the canine T2R5, (C) a close-up view of selected residues lining theactive site pocket interacting with, or close to, 1, 10 Phenanthroline,(D) a ligand interaction map demonstrating potential interaction sitesbetween 1,10 Phenanthroline and T2R5 and (E) a dose-response curve for1, 10 Phenanthroline when tested against canine T2R5 in vitro. There isa potential hydrogen bond between Ser89 and each nitrogen of 1, 10Phenanthroline. Additional residues that can potentially make van derWaals or Pi interactions with the ligand include Pro264, Leu58, Val88,Gln90, Ile86, Leu173, Trp165, Thr258, Ala261, Tyr234, Glu257, Met260,and Trp85.

FIG. 8A-E shows (A) the chemical structure of Cucurbitacin B, (B) insilico modeling of Cucurbitacin B docked within the active site of thecanine T2R10, (C) a close-up view of selected residues lining the activesite pocket interacting with, or close to, Cucurbitacin B, (D) a ligandinteraction map demonstrating potential interaction sites betweenCucurbitacin B and T2R10 and (E) a dose-response curve for CucurbitacinB when tested against canine T2R10 in vitro. Lys258 and Leu180(backbone) can potentially make hydrogen bonds with the ligand.Additional residues that can potentially make polar, hydrogen bonding,pi interactions, or charged interactions with the ligand include Lys170,Glu172, and Asn181. Residues that can potentially make van der Waalsinteractions with the ligand include Phe261, Met265, Ile262, Gln169,Lys69, Met168, Ile245, Val90, Phe242, Gln94, Val184, Asn93, Trp89, andTyr241.

FIG. 9A-E shows (A) the chemical structure of Propylthiouracil, (B) insilico modeling of Propylthiouracil docked within the active site of thecanine T2R43, (C) a close-up view of selected residues lining the activesite pocket interacting with, or close to, Propylthiouracil, (D) aligand interaction map demonstrating potential interaction sites betweenPropylthiouracil and T2R43 and (E) a dose-response curve forPropylthiouracil when tested against canine T2R43 in vitro. Residuesthat can potentially make hydrogen bond or charged interactions with theligand include Tyr241, Trp88, and Thr181. Additional residues that canpotentially make polar, hydrogen bonding, pi interactions, or chargedinteractions with the ligand include Met177, Asn92, Asn184, and Phe185.Additional residues that can potentially make van der Waals interactionswith the ligand include Gln152, His143, Phe261, Ala172, His85, Asp170,Lys265, Phe242, Leu245, Thr89, and Phe180.

FIG. 10 shows a summary table of receptor-ligand interactions detailedin FIGS. 3-9. (+) indicates that the ligand elicited a clear dosedependent response from the receptor in vitro; (−) indicates that theligand did not elicit a response specific, dose dependent response fromthe receptor in vitro; and shaded cells indicate the interactionsdetailed in FIGS. 3-9.

DETAILED DESCRIPTION

The presently disclosed subject matter relates to methods for screeningand identifying compounds that modulate the activity and/or expressionof bitter taste receptors. The presently disclosed subject matterfurther relates to making palatable, nutritionally-complete pet foodproducts and medicines, wherein the raw materials of the pet food and/orfinalized pet food product or medicine is screened to determine if itcontains compounds that modulate the bitter taste receptors.Furthermore, such screening methods can be used to select raw materialsand/or finalized pet food products that do not comprise bitter receptoractivating compounds. Compounds identified through said methods can beused to modify the palatability of pet food products and medicines byincreasing or decreasing a bitter taste. Said compounds can also be usedto increase a bitter taste of an object, and thereby reduce palatabilityand ingestion by a dog.

1. Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the methods and compositions of theinvention and how to make and use them.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Still further, the terms“having,” “including,” “containing” and “comprising” are interchangeableand one of skill in the art is cognizant that these terms are open endedterms.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 3 or more than 3 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. As used herein, “taste” refers toa sensation caused by activation of receptor cells in a subject's tastebuds. In certain embodiments, taste can be selected from the groupconsisting of sweet, sour, salt, bitter, kokumi and umami. In certainembodiments, “taste” can include free fatty acid taste. See, e.g.,Cartoni et al., J. of Neuroscience, 30(25): 8376-8382 (2010), thecontents of which are incorporated herein by reference. In certainembodiments, a taste is elicited in a subject by a “tastant.” In certainembodiments, a tastant can be a synthetic tastant. In certainembodiments, the tastant is obtained or prepared from a natural source.

As used herein, “taste profile” refers to a combination of tastes, suchas, for example, one or more of a sweet, sour, salt, bitter, umami,kokumi and free fatty acid taste. In certain embodiments, a tasteprofile is produced by one or more tastant that is present in acomposition at the same or different concentrations. In certainembodiments, a taste profile refers to the intensity of a taste orcombination of tastes, for example, a sweet, sour, salt, bitter, umami,kokumi and free fatty acid taste, as detected by a subject or any assayknown in the art. In certain embodiments, modifying, changing or varyingthe combination of tastants in a taste profile can change the sensoryexperience of a subject.

As used herein, “flavor” refers to one or more sensory stimuli, such as,for example, one or more of taste (gustatory), smell (olfactory), touch(tactile) and temperature (thermal) stimuli. In certain non-limitingembodiments, the sensory experience of a subject exposed to a flavor canbe classified as a characteristic experience for the particular flavor.For example, a flavor can be identified by the subject as being, but notlimited to, a floral, citrus, berry, nutty, caramel, chocolate, peppery,smoky, cheesy, meaty, etc., flavor. As used herein, a flavor compositioncan be selected from a liquid, solution, dry powder, spray, paste,suspension and any combination thereof. The flavor can be a naturalcomposition, an artificial composition, a nature identical, or anycombination thereof.

As used interchangeably herein, “aroma” and “smell” refer to anolfactory response to a stimulus. For example, and not by way oflimitation, an aroma can be produced by aromatic substances that areperceived by the odor receptors of the olfactory system.

As used herein, “flavor profile” refers to a combination of sensorystimuli, for example, tastes, such as sweet, sour, bitter, salty, umami,kokumi and free fatty acid tastes, and/or olfactory, tactile and/orthermal stimuli. In certain embodiments, the flavor profile comprisesone or more flavors which contribute to the sensory experience of asubject. In certain embodiments, modifying, changing or varying thecombination of stimuli in a flavor profile can change the sensoryexperience of a subject.

As used herein “admixing,” for example, “admixing the flavor compositionor combinations thereof of the present application with a food product,”refers to the process where the flavor composition, or individualcomponents of the flavor composition, is mixed with or added to thecompleted product or mixed with some or all of the components of theproduct during product formation or some combination of these steps.When used in the context of admixing, the term “product” refers to theproduct or any of its components. This admixing step can include aprocess selected from the step of adding the flavor composition to theproduct, spraying the flavor composition on the product, coating theflavor composition on the product, suspending the product in the flavorcomposition, painting the flavor composition on the product, pasting theflavor composition on the product, encapsulating the product with theflavor composition, mixing the flavor composition with the product andany combination thereof. The flavor composition can be a solution,liquid, dry powder, spray, paste, suspension and any combinationthereof.

As used herein, “palatability” can refer to the overall willingness of ahuman or non-human animal, for example, a companion animal, to eat acertain food product. Increasing the “palatability” of a food productcan lead to an increase in the enjoyment and acceptance of the food bythe human or non-human animal to ensure the human or non-human animaleats a “healthy amount” of the food. Decreasing the “palatability” of afood product can lead to a decrease in the enjoyment and acceptance ofthe food by the human or non-human animal. The term “healthy amount” ofa food as used herein refers to an amount that enables the human ornon-human animal to maintain or achieve an intake contributing to itsoverall general health in terms of micronutrients, macronutrients andcalories, for example, such as set out in the “Mars Petcare EssentialNutrient Standards.” In certain embodiments, “palatability” can mean arelative preference of a human or non-human animal for one food productover another. For example, when a human or non-human animal shows apreference for one of two or more food products, the preferred foodproduct is more “palatable,” and has “enhanced palatability.” In certainembodiments, the relative palatability of one food product compared toone or more other food products can be determined, for example, inside-by-side, free-choice comparisons, e.g., by relative consumption ofthe food products, or other appropriate measures of preferenceindicative of palatability. Palatability can be determined by a standardtesting protocol in which the animal has equal access to both foodproducts such as a test called “two-bowl test” or “versus test.” Suchpreference can arise from any of the animal's senses, but can be relatedto, inter alia, taste, aftertaste, smell, mouth feel and/or texture.

The term “pet food” or “pet food product” or “final pet food product”means a product or composition that is intended for consumption by acompanion animal, such as cats, dogs, guinea pigs, rabbits, birds andhorses. For example, but not by way of limitation, the companion animalcan be a “domestic” dog, e.g., Canis lupus familiaris. In certainembodiments, the companion animal can be a “domestic” cat such as Felisdomesticus. A “pet food” or “pet food product” includes any food, feed,snack, food supplement, liquid, beverage, treat, toy (chewable and/orconsumable toys), meal substitute or meal replacement.

The term “human food” or “human food product” or “final human foodproduct” means a product or composition that is intended for consumptionby a human. A “human food” or “human food product” includes any food,feed, snack, food supplement, liquid, beverage, treat, meal substituteor meal replacement.

In certain embodiments, a “food product” includes human and/or pet foodproducts.

As used herein “nutritionally-complete” refers to pet food product thatcontains all known required nutrients for the intended recipient of thepet food product, in appropriate amounts and proportions based, forexample, on recommendations of recognized or competent authorities inthe field of companion animal nutrition. Such foods are thereforecapable of serving as a sole source of dietary intake to maintain life,without the addition of supplemental nutritional sources.

The term “raw material” means a plant and/or animal material beforebeing processed or manufactured into a final pet food product. Incertain embodiments, a “raw material” is not significantly processed inorder to separate it into individual elements prior to analysis (e.g.,by extraction, purification, fractionation and/or concentration). A “rawmaterial” includes a protein source for a pet food product. In certainembodiments, the raw material is a novel protein source that does notcompete with the human food sources (i.e., a protein source that is notcommonly eaten by humans). In certain embodiments, the raw material is aby-product of the human food chain. In certain non-limiting embodiments,the “raw material” is processed, for example, in order to separate itinto individual elements prior to analysis (e.g., by extraction,purification, fractionation and/or concentration), prior to beinganalyzed according to the methods described herein.

As used herein “flavor composition” refers to at least one compound orbiologically acceptable salt thereof that modulates, includingenhancing, multiplying, potentiating, decreasing, suppressing, orinducing, the tastes, smells, flavors and/or textures of a natural orsynthetic tastant, flavoring agent, taste profile, flavor profile and/ortexture profile in an animal or a human. In certain embodiments, theflavor composition comprises a combination of compounds or biologicallyacceptable salts thereof. In certain embodiments, the flavor compositionincludes one or more excipients.

As used herein, “taste deterrent,” “taste deterrent product,” or “tastedeterrent composition” refers to a product or composition containing atleast one compound or biologically acceptable salt thereof that providesa bitter taste to an object. In certain embodiments, the taste deterrentdiscourages an animal from chewing, licking, or consuming an object, forexample, a food or liquid product. In certain embodiments, the objectis, for example but not limited to, clothing, shoes, carpet, furniture,household items, pesticides, herbicides, or poisonous compounds. Incertain embodiments, the object is another animal or the animal itself.In other embodiment, the object is toxic to the animal, or would bedetrimental to the animal's health upon ingestion.

As used herein, the terms “modulates” or “modifies” refers to anincrease or decrease in the amount, quality or effect of a particularactivity of a receptor and/or an increase or decrease in the expression,activity or function of a receptor. “Modulators,” as used herein, referto any inhibitory or activating compounds identified using in silico, invitro and/or in vivo assays for, e.g., agonists, antagonists, allostericmodulators and their homologs, including fragments, variants andmimetics.

“Inhibitors” or “antagonists,” as used herein, refer to modulatingcompounds that reduce, decrease, block, prevent, delay activation,inactivate, desensitize or down regulate the biological activity and/orexpression of a receptor or pathway of interest.

The term “antagonist” includes full, partial, and neutral antagonists aswell as inverse agonists.

“Inducers,” “activators” or “agonists,” as used herein, refer tomodulating compounds that increase, induce, stimulate, open, activate,facilitate, enhance activation, sensitize or upregulate a receptor orpathway of interest. The term “agonist” includes full and partialagonists.

“Allosteric modulators” as used herein, refer to “positive allostericmodulators” and “negative allosteric modulators.” “Positive allostericmodulators” refer to modulating compounds that increase, induce,stimulate, open, activate, facilitate, enhance activation, sensitize orup regulate a receptor or pathway of interest caused by the binding of adifferent compound to the receptor. “Negative allosteric modulators”refer to modulating compounds that reduce, decrease, block, prevent,delay activation, inactivate, desensitize or down regulate thebiological activity and/or expression of a receptor or pathway ofinterest caused by the binding of a different compound to the receptor.

As used herein, the terms “vector” and “expression vector” refer to DNAmolecules that are either linear or circular, into which another DNAsequence fragment of appropriate size can be integrated. Such DNAfragment(s) can include additional segments that provide fortranscription of a gene encoded by the DNA sequence fragment. Theadditional segments can include and are not limited to:

promoters, transcription terminators, enhancers, internal ribosome entrysites, untranslated regions, polyadenylation signals, selectablemarkers, origins of replication and such like. Expression vectors areoften derived from plasmids, cosmids, viral vectors and yeast artificialchromosomes. Vectors are often recombinant molecules containing DNAsequences from several sources.

The term “operably linked,” when applied to DNA sequences, for examplein an expression vector, indicates that the sequences are arranged sothat they function cooperatively in order to achieve their intendedpurposes, i.e., a promoter sequence allows for initiation oftranscription that proceeds through a linked coding sequence as far asthe termination signal.

The term “nucleic acid molecule” and “nucleotide sequence,” as usedherein, refers to a single or double stranded covalently-linked sequenceof nucleotides in which the 3′ and 5′ ends on each nucleotide are joinedby phosphodiester bonds. The nucleic acid molecule can includedeoxyribonucleotide bases or ribonucleotide bases, and can bemanufactured synthetically in vitro or isolated from natural sources.

The terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,”used interchangeably herein, refer to a molecule formed from the linkingof at least two amino acids. The link between one amino acid residue andthe next is an amide bond and is sometimes referred to as a peptidebond. A polypeptide can be obtained by a suitable method known in theart, including isolation from natural sources, expression in arecombinant expression system, chemical synthesis or enzymaticsynthesis. The terms can apply to amino acid polymers in which one ormore amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymers.

The term “amino acid,” as used herein, refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, gamma-carboxyglutamate and O-phosphoserine. Aminoacid analogs and derivatives can refer to compounds that have the samebasic chemical structure as a naturally occurring amino acid, i.e., acarbon that is bound to a hydrogen, a carboxyl group, an amino group andan R group, e.g., homoserine, norleucine, methionine sulfoxide andmethionine methyl sulfonium. Such analogs can have modified R groups(e.g., norleucine) or modified peptide backbones, but retain the samebasic chemical structure as a naturally occurring amino acid. Amino acidmimetics means chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunction in a manner similar to a naturally occurring amino acid.

The terms “isolated” or “purified”, used interchangeably herein, refersto a nucleic acid, a polypeptide, or other biological moiety that isremoved from components with which it is naturally associated. The term“isolated” can refer to a polypeptide that is separate and discrete fromthe whole organism with which the molecule is found in nature or ispresent in the substantial absence of other biological macromolecules ofthe same type. The term “isolated” with respect to a polynucleotide canrefer to a nucleic acid molecule devoid, in whole or part, of sequencesnormally associated with it in nature; or a sequence, as it exists innature, but having heterologous sequences in association therewith; or amolecule disassociated from the chromosome.

As used herein, the term “recombinant” can be used to describe a nucleicacid molecule and refers to a polynucleotide of genomic, RNA, DNA, cDNA,viral, semisynthetic or synthetic origin which, by virtue of its originor manipulation is not associated with all or a portion of thepolynucleotide with which it is associated in nature.

The term “fusion,” as used herein, refers to joining of differentpeptide or protein segments by genetic or chemical methods wherein thejoined ends of the peptide or protein segments may be directly adjacentto each other or may be separated by linker or spacer moieties such asamino acid residues or other linking groups.

2. Bitter Taste Receptors

The presently disclosed subject matter provides bitter taste receptorsfor use in the disclosed methods. The bitter taste receptors of thepresent disclosure can include mammalian bitter taste receptors such as,but not limited to, canine bitter taste receptors.

In certain non-limiting embodiments, the bitter taste receptor is acanine bitter taste receptor, for example, canine bitter taste receptorT2R1, T2R2, T2R3, T2R4, T2R5, T2R7, T2R10, T2R12, T2R38, T2R39, T2R40,T2R41, T2R42, T2R43, T2R62, T2R67, or combinations thereof.

In certain embodiments, a bitter taste receptor for use in the presentlydisclosed methods encompasses a canine bitter taste receptor having thenucleotide sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 and/or the amino acid sequence set forthin SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, or 32, including fragments thereof (e.g., functional fragmentsthereof) and variants thereof.

In certain non-limiting embodiments, a bitter taste receptor for use inthe presently disclosed methods does not include a feline bitter tastereceptor.

In certain embodiments, the bitter taste receptor for use in thepresently disclosed subject matter can include a receptor encoded by anucleotide sequence that is at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% homologous to any one of SEQ IDNOs:1-16 (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor for use in thepresently disclosed methods can include a receptor comprising an aminoacid sequence that is between about 33 and 99%, between about 34 and99%, between about 35 and 99%, between about 40 and 99%, between about45 and 99%, between about 50 and 99%, between about 55 and 99%, betweenabout 60 and 99%, between about 61 and 99%, between about 65 and 99%,between about 70 and 99%, between about 72 and 99%, between about 75 and99%, between about 79 and 99%, between about 80 and 99%, between about84 and 99%, between about 85 and 99%, between about 87 and 99%, betweenabout 89 and 99%, between about 90 and 99%, between about 95 and 99%, orbetween about 97 and 99% homologous to any one of SEQ ID NOs:17-32(homology, as that term is used herein, may be measured using standardsoftware such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor for use in thepresently disclosed methods can include a receptor comprising an aminoacid sequence that is at least about 33%, 34%, 35%, 40%, 45%, 50%, 55%,60%, 61%, 65%, 70%, 72%, 75%, 79%, 80%, 84%, 85%, 87%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to any one of SEQID NOs:17-32 (homology, as that term is used herein, may be measuredusing standard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R1comprising an amino acid sequence as set forth in SEQ ID NO:17, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:1, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R2comprising an amino acid sequence as set forth in SEQ ID NO:18, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:2, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R3comprising an amino acid sequence as set forth in SEQ ID NO:19, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:3, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R4comprising an amino acid sequence as set forth in SEQ ID NOs:20, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:4, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R5comprising an amino acid sequence as set forth in SEQ ID NO:21, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:5, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R7comprising an amino acid sequence as set forth in SEQ ID NO:22, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:6, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R10comprising an amino acid sequence as set forth in SEQ ID NO:23, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:7, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R12comprising an amino acid sequence as set forth in SEQ ID NO:24, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:8, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R38comprising an amino acid sequence as set forth in SEQ ID NO:25, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:9, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R39comprising an amino acid sequence as set forth in SEQ ID NO:26, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:10, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R40comprising an amino acid sequence as set forth in SEQ ID NO:27, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:11, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R41comprising an amino acid sequence as set forth in SEQ ID NO: 28, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO: 12,or a sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percenthomologous thereto (homology, as that term is used herein, may bemeasured using standard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R42comprising an amino acid sequence as set forth in SEQ ID NO: 29, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO: 13,or a sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percenthomologous thereto (homology, as that term is used herein, may bemeasured using standard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R43comprising an amino acid sequence as set forth in SEQ ID NO: 30, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO: 14,or a sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percenthomologous thereto (homology, as that term is used herein, may bemeasured using standard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R62comprising an amino acid sequence as set forth in SEQ ID NO: 31, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO:15, ora sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA).

In certain embodiments, the bitter taste receptor is a canine T2R67comprising an amino acid sequence as set forth in SEQ ID NO: 32, or asequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologousthereto (homology, as that term is used herein, may be measured usingstandard software such as BLAST or FASTA), and is encoded, for example,by a nucleic acid comprising a sequence as set forth in SEQ ID NO: 16,or a sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percenthomologous thereto (homology, as that term is used herein, may bemeasured using standard software such as BLAST or FASTA).

In certain embodiments, homology is described as a percent identitybetween two sequences. The percent identity of two amino acid sequencesor of two nucleotide sequences can be determined by aligning thesequences for optimal comparison purposes (e.g., gaps can be introducedin the first sequence for best alignment with the sequence) andcomparing the amino acid residues or nucleotides at correspondingpositions. The percent identity can be determined by the number ofidentical amino acid residues or nucleotides in the sequences beingcompared (e.g., % identity=number of identical positions/total number ofpositions ×100).

The determination of percent identity between two sequences can bedetermined using a mathematical algorithm known to those of skill in theart. A non-limiting example of a mathematical algorithm for comparingtwo sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877, the disclosures of which areincorporated herein by reference in their entireties. The NBLAST andXBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410have incorporated such an algorithm. BLAST nucleotide searches can beperformed with the NBLAST program, for example, score=100,wordlength=12, to obtain nucleotide sequences homologous to nucleotidesequences of the invention. BLAST protein searches can be performed withthe XBLAST program, for example, score=50, wordlength=3, to obtain aminoacid sequences homologous to amino acid sequence of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997) Nucleic Acids Res.25:3389-3402, the disclosure of which is incorporated herein byreference in its entirety. Alternatively, PSI-Blast can be used toperform an iterated search, which detects distant relationships betweenmolecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) can be used. See http://www.ncbi.nlm.nih.gov. An additionalnon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989), the disclosure of which is incorporated herein by reference inits entirety. The ALIGN program (version 2.0), which is part of the CGCsequence alignment software package, has incorporated such an algorithm.Other non-limiting examples of algorithms for sequence analysis known inthe art include ADVANCE and ADAM as described in Torellis and Robotti(1994) Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson andLipman (1988) Proc. Natl. Acad. Sci. 85:2444-8, the disclosures of whichare incorporated herein by reference in their entireties. Within FASTA,ktup is a control option that sets the sensitivity and speed of thesearch.

In certain embodiments, the disclosed subject matter provides for theuse of an isolated or purified bitter taste receptor and/or variants andfragments thereof. The disclosed subject matter also encompasses the useof sequence variants. In certain embodiments, variation can occur ineither or both the coding and non-coding regions of a nucleotidesequence of a bitter taste receptor. Variants can include asubstantially homologous protein encoded by the same genetic locus in anorganism, i.e., an allelic variant. Variants also encompass proteinsderived from other genetic loci in an organism, e.g., canine, but havingsubstantial homology to the bitter taste receptor, i.e., a homolog.Variants can also include proteins substantially homologous to thebitter taste receptor but derived from another organism, i.e., anortholog. Variants also include proteins that are substantiallyhomologous to the bitter taste receptor that are produced by chemicalsynthesis. Variants also include proteins that are substantiallyhomologous to the bitter taste receptor that are produced by recombinantmethods.

Orthologs, homologs and allelic variants can be identified using methodswell known in the art. These variants can include a nucleotide sequenceencoding a receptor that is at least about 60-65%, about 65-70%, about70-75, about 80-85%, about 90-95%, about 95-99% or more homologous tothe nucleotide sequence shown in any one of SEQ ID NOs: 1-16, orfragments thereof. Such nucleic acid molecules can readily be identifiedas being able to hybridize under stringent conditions, to the nucleotidesequence shown in any one of SEQ ID NOs:1-16, or a fragment thereof Incertain embodiments, two polypeptides (or regions thereof) aresubstantially homologous when the amino acid sequences are at leastabout 60-65%, about 65-70%, about 70-75, about 80-85%, about 90-95%,about 95-99% or more homologous to the amino acid sequences shown in anyone of SEQ ID NOs: 17-32, or a fragment thereof. A substantiallyhomologous amino acid sequence, according to the disclosed subjectmatter, will be encoded by a nucleic acid sequence hybridizing to thenucleic acid sequence, or portion thereof, of the nucleotide sequenceshown in any one of SEQ ID NOs: 1-16 under stringent conditions.

The bitter taste receptors for use in the methods of the disclosedsubject matter include bitter taste receptors having additions,deletions or substitutions of amino acid residues (variants) which donot substantially alter the biological activity of the receptor. Thoseindividual sites or regions of the bitter taste receptors which may bealtered without affecting biological activity can be determined byexamination of the structure of the bitter taste receptor extracellulardomain, for example. Alternatively and/or additionally, one canempirically determine those regions of the receptor which would tolerateamino acid substitutions by alanine scanning mutagenesis (Cunningham etal., Science 244, 1081-1085 (1989), the disclosure of which is herebyincorporated by reference in its entirety). In the alanine scanningmutagenesis method, selected amino acid residues are individuallysubstituted with a neutral amino acid (e.g., alanine) in order todetermine the effects on biological activity.

It is generally recognized that conservative amino acid changes areleast likely to perturb the structure and/or function of a polypeptide.Accordingly, the disclosed subject matter encompasses one or moreconservative amino acid changes within a bitter taste receptor.Conservative amino acid changes generally involve substitution of oneamino acid with another that is similar in structure and/or function(e.g., amino acids with side chains similar in size, charge and shape).Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). In certainembodiments, one or more amino acid residues within a bitter tastereceptor can be replaced with other amino acid residues from the sameside chain family and the altered protein can be tested for retainedfunction using the functional assays described herein. Modifications canbe introduced into a bitter taste receptor of the present disclosure bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis. If such substitutions result in aretention in biological activity, then more substantial changes can beintroduced and/or other additions/deletions may be made and theresulting products screened. In certain embodiments, deletions oradditions can be from 5-10 residues, alternatively from 2-5 amino acidresidues or from 1-2 residues, and values in between.

The disclosed subject matter also provides for fusion proteins thatcomprise a bitter taste receptor, or fragment thereof. In certainembodiments, the disclosed subject matter provides for fusion proteinsof a bitter taste receptor, or functional fragments thereof, and animmunoglobulin heavy chain constant region. In certain embodiments, afusion protein of the present disclosure can include a detectablemarker, a functional group such as a carrier, a label, a stabilizingsequence or a mechanism by which bitter taste receptor agonist bindingcan be detected. Non-limiting embodiments of a label include a FLAG tag,a His tag, a MYC tag, a maltose binding protein and others known in theart. The presently disclosed subject matter also provides nucleic acidsencoding such fusion proteins, vectors containing fusionprotein-encoding nucleic acids and host cells comprising such nucleicacids or vectors. In certain embodiments, fusions can be made at theamino terminus (N-terminus) of a bitter taste receptor or at the carboxyterminus (C-terminus) of a bitter taste receptor.

In certain embodiments, the bitter taste receptors disclosed herein cancontain additional amino acids at the N-terminus and/or at theC-terminus end of the sequences, e.g., when used in the methods of thedisclosed subject matter. In certain embodiments, the additional aminoacids can assist with immobilizing the polypeptide for screeningpurposes, or allow the polypeptide to be part of a fusion protein, asdisclosed above, for ease of detection of biological activity.

3. Methods for Identifying Bitter Taste Receptor Modulating Compounds

The present disclosure further provides methods for identifyingcompounds that modulate the activity and/or expression of a bitter tastereceptor. For example, and not by way of limitation, the modulator canbe an agonist (for example, a full or partial agonist), or anantagonist, or an inverse agonist, or an allosteric modulator. Thepresently disclosed subject matter provides in silico and in vitromethods for identifying compounds that modulate the activity and/orexpression of a bitter taste receptor, disclosed above.

3.1 In silico Methods

The presently disclosed subject matter further provides in silicomethods for identifying compounds that can potentially interact with abitter taste receptor and/or modulate the activity and/or expression ofa bitter taste receptor.

In certain embodiments, the method can include predicting thethree-dimensional structure (3D) of a bitter taste receptor andscreening the predicted 3D structure with putative bitter taste receptormodulating compounds (i.e., test compounds). The method can furtherinclude predicting whether the putative compound would interact with thebinding site of the receptor by analyzing the potential interactionswith the putative compound and the amino acids of the receptor. Themethod can further include identifying a test compound that can bind toand/or modulate the biological activity of the bitter taste receptor bydetermining whether the 3D structure of the compound fits within thebinding site of the 3D structure of the receptor.

In certain embodiments, the bitter taste receptor for use in thedisclosed method can be a canine T2R1, T2R2, T2R3, T2R4, T2R5, T2R7,T2R10, T2R12, T2R38, T2R39, T2R40, T2R41, T2R42, T2R43, T2R62, T2R67, orcombinations thereof.

In other embodiments, the bitter taste receptor for use in the disclosedmethod can have the amino acid sequence of any one of SEQ ID NO:17-32,or a fragment or variant thereof. In certain embodiments, the bittertaste receptor for use in the presently disclosed subject matter caninclude a receptor comprising an amino acid sequence having at leastabout 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 65%, 70%, 72%, 75%,79%, 80%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity to any one of SEQ ID NO:17-32, or a fragment orvariant thereof. In certain embodiments, the bitter taste receptor foruse in the disclosed method can be encoded by a nucleotide sequence ofany one of SEQ ID NO: 1-16, or a fragment or variant thereof. In certainembodiments, the bitter taste receptor for use in the presentlydisclosed subject matter can include a receptor encoded by a nucleotidesequence having at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identity to any one of SEQ ID NO:1-16, or afragment or variant thereof.

Non-limiting examples of compounds (e.g., potential bitter tastereceptor modulators) that can be tested using the disclosed methodsinclude any small chemical compound, or any biological entity, such aspeptides, salts, amino acids and bitter compound known in the art, e.g.denatonium benzoate. In certain embodiments, the test compound can be asmall chemical molecule.

In certain embodiments, structural models of a bitter taste receptor canbe built using crystal structures of other GPCRs as templates forhomology modeling. For example, and not by way of limitation, structuralmodels can be generated using the crystal structures of Group 1 GPCRs.Bitter receptors belong to a separate subclass of GPCR's for whichcrystal structures have not been solved yet. In certain embodiments, astructural model of a bitter taste receptor can be based on a known or acombination of known crystal structures of GPCRs. (See, e.g., Lee etal., Eur J Pharmacol. 2015 May 14. pii: S0014-2999(15)30012-1, which isincorporated by reference in its entirety herein). In certainembodiments, a structural model of a bitter taste receptor can begenerated based on the crystal structure of a β2 adrenergic receptor,3SN6 from Protein Data Bank (PDB). (See, e.g., Rasmussen et al., Nature.2011 Jul 19;477(7366):549-55, which is incorporated by reference in itsentirety herein). In certain embodiments, a structural model of the 7transmembrane domain (7TM) of a bitter taste receptor can be generatedbased on the crystal structures of existing GPCR crystal structure 3SN6from PDB.

Any suitable modeling software known in the art can be used. In certainembodiments, the Modeller software package can be used to generate thethree-dimensional protein structure.

In certain embodiments, the in silico methods of identifying a compoundthat binds to a T2R comprises determining whether a test compoundinteracts with one or more amino acids of a T2R binding pocket, asdescribed herein.

Compounds that are identified by the disclosed in silico methods can befurther tested using the in vitro and in vivo methods disclosed herein.

3.2 T2R Transmembrane Compound Binding Site

The present application provides for methods of screening for compoundsthat modulate the activity of a bitter taste receptor, for example, acanine T2R receptor, wherein the compounds interact with one or moreamino acids of the bitter taste receptor. In certain embodiments, thebinding site of a bitter taste receptor comprises amino acids within the7TM domain of the receptor, and can be identified by generating aninteraction map of the receptor using in silico modeling, as describedherein. In one non-limiting example, the presence of an amino acid inthe 7TM interaction map means that the residue is in the vicinity of theligand binding environment, an interacts with the ligand.

In certain embodiments, the interaction between an amino acid in the 7TMinteraction map and the ligand is a pi-pi interaction.

In certain embodiments, the interaction between an amino acid in the 7TMinteraction map and the ligand is a hydrogen bond interaction.

In certain embodiments, the interaction between an amino acid in the 7TMinteraction map and the ligand is a hydrophobic interaction.

In certain embodiments, the interaction between an amino acid in the 7TMinteraction map and the ligand is a van de Waals interaction.

In certain embodiments, the amino acid in the 7TM interaction map is apolar amino acid, wherein the amino acid interacts with the ligand as ahydrogen bond donor and/or acceptor.

In certain embodiments, the interaction between a compound and one ormore amino acids of the T2R receptors described herein can comprises oneor more hydrogen bond, covalent bond, non-covalent bond, salt bridge,physical interaction, and combinations thereof. The interactions canalso be any interaction characteristic of a ligand receptor interactionknown in the art. Such interactions can be determined by, for example,site directed mutagenesis, x-ray crystallography, x-ray or otherspectroscopic methods, Nuclear Magnetic Resonance (NMR), cross-linkingassessment, mass spectroscopy or electrophoresis, cryo-microscopy,displacement assays based on known agonists, structural determinationand combinations thereof.

In certain embodiments, the interactions are determined in silico, forexample, by theoretical means such as docking a compound into a canineT2R binding pocket using molecular docking, molecular modeling,molecular simulation, or other means known to persons of ordinary skillin the art. In certain embodiments, the T2R receptor is a canine T2R,for example, but not limited to, T2R1, T2R2, T2R3, T2R4, T2R5, T2R7,T2R10, T2R12, T2R38, T2R39, T2R40, T2R41, T2R42, T2R43, T2R62, andT2R67.

In certain embodiments, the T2R is a T2R present in canine but notpresent in feline animals, for example, T2R5, T2R39, T2R40, T2R41,and/or T2R62.

In certain embodiments, the compounds interact with one or more T2Rreceptors described herein according to any combination of interactionsdescribed herein, for example, one, two, three or more of theinteractions.

In certain embodiments, the compounds bind to at least one of thereceptors described herein. In certain embodiment, the compounds bindselectively to only one of the receptors described herein.

In one embodiment, the bitter taste receptor is a canine T2R1. Incertain embodiments, the amino acids that the compounds interact withcomprise Asn89 and/or Tyr239, for example, by polar or hydrogen bonding,as exemplified by in silico modeling of Menthol in T2R1 (FIG. 3).Alternatively, or in addition, in certain embodiments, the amino acidsthat the compounds interact with comprise any one, two, three or more ofthe T2R1 residues Asn89, Tyr239, Ile167, Gln174, Glu169, Phe257, Ala242,Phe177, His238, Cys260, Phe264, Leu234, Cys235, Phe85, Leu261, Leu178,Leu181, Val86, and Phe82, for example, by polar, hydrogen bond, saltbridge, van der Waals, pi, or other interactions, as exemplified by insilico modeling of Menthol in T2R1 (FIG. 3).

In one embodiment, the bitter taste receptor is a canine T2R2, which isshared by dogs and cats, but not humans, where it is a pseudogene. Incertain embodiments, the amino acids that the compounds interact withcomprise one or more of T2R2 residues Ser94, Trp90, Lys268, Tyr245,and/or Glu180, for example, by hydrogen bonding or salt bridgeinteractions, as exemplified by in silico modeling of Ofloxacin in T2R2(FIG. 4). Alternatively, or in addition, in certain embodiments, theamino acids that the compounds interact with comprise T2R2 residuesArg176 and/or Met91, either alone or in conjunction with interactionslisted above, for example, by polar, hydrogen bonding, or chargedinteractions, as exemplified by in silico modeling of Ofloxacin in T2R2(FIG. 4). Alternatively, or in addition, in certain embodiments, theamino acids that the compounds interact with comprise any one, two,three or more of the T2R2 residues Ser94, Trp90, Lys268, Tyr245, Glu180,Arg176, Met91, Asn185, Val184, Met181, Phe249, Pro155, Gln177, Lys174,Phe264, Phe93, Leu59, Met271, Phe246, and Leu188, for example, by polar,hydrogen bond, salt bridge, van der Waals, pi, or other interactions, asexemplified by in silico modeling of Ofloxacin in T2R2 (FIG. 4).

In one embodiment, the bitter taste receptor is a canine T2R3. Incertain embodiments, the amino acids that the compounds interact withcomprise T2R3 residues Asn93 and/or Asp86, for example, by hydrogenbonding or salt bridge interactions, as exemplified by in silicomodeling of Chloroquine in T2R3 (FIG. 5). Alternatively, or in addition,in certain embodiments, the amino acids that the compounds interact withcomprise any one or more of the T2R3 residues Tyr246, Phe247, Thr186,Asn189, Trp89, Asp86, and Arg175, either alone or in conjunction withinteractions listed above, for example, by polar, hydrogen bonding, orcharged interactions, as exemplified by in silico modeling ofChloroquine in T2R3 (FIG. 5). Alternatively, or in addition, in certainembodiments, the amino acids that the compounds interact with compriseany one, two, three or more of the T2R3 residues Asn93, Asp86, Tyr246,Phe247, Thr186, Asn189, Trp89, Asp86, Arg175, Phe250, Glyl85, Phe243,Thr90, Asn176, Val149, Ile154, Lys174, Met82, Ile85, Lys173, and Met69,for example, by polar, hydrogen bond, salt bridge, van der Waals, pi, orother interactions, as exemplified by in silico modeling of Chloroquinein T2R3 (FIG. 5).

In one embodiment, the bitter taste receptor is a canine T2R4. Incertain embodiments, the amino acids that the compounds interact withcomprise any one or more of T2R4 residues Ser186, Asp93, and Tyr240, forexample, by hydrogen bonding or salt bridge interactions, as exemplifiedby in silico modeling of Colchicine in T2R4 (FIG. 6). Alternatively, orin addition, in certain embodiments, the amino acids that the compoundsinteract with comprise any one or more of T2R4 residues Ser94, Leu97,Asn95, Leu92, Ser96, Trp98, Val187, and Thr247, either alone or inconjunction with interactions listed above, for example, by polar,hydrogen bonding, or charged interactions, as exemplified by in silicomodeling of Colchicine in T2R4 (FIG. 6). Alternatively, or in addition,in certain embodiments, the amino acids that the compounds interact withcomprise any one, two, three or more of the T2R4 residues Ser186, Asp93,Tyr240, Ser94, Leu97, Asn95, Leu92, Ser96, Trp98, Val187, Thr247,Tyr243, Trp89, Met58, Ser269, Pro273, Ser270, Gln189, Thr144, Leu188,Val183, Leu182, Ser244, and Met90, for example, by polar, hydrogen bond,salt bridge, van der Waals, pi, or other interactions, as exemplified byin silico modeling of Colchicine in T2R4 (FIG. 6).

In one embodiment, the bitter taste receptor is a canine T2R5, which ispresent in dogs and humans, but not cats. In certain embodiments, theamino acids that the compounds interact with comprise T2R5 residueSer89, for example, by hydrogen bonding or salt bridge interactions, asexemplified by in silico modeling of 1,10 Phenanthroline in T2R5 (FIG.7). Alternatively, or in addition, in certain embodiments, the aminoacids that the compounds interact with comprise any one, two, three ormore of the T2R5 residues Ser89, Pro264, Leu58, Val88, Gln90, Ile86,Leu173, Trp165, Thr258, Ala261, Tyr234, Glu257, Met260, and Trp85, forexample, by polar, hydrogen bond, salt bridge, van der Waals, pi, orother interactions, as exemplified by in silico modeling of 1,10Phenanthroline in T2R5 (FIG. 7).

In one embodiment, the bitter taste receptor is a canine T2R10. Incertain embodiments, the amino acids that the compounds interact withcomprise T2R10 residues Lys258 and/or Leu180 (backbone), for example, byhydrogen bonding or salt bridge interactions, as exemplified by insilico modeling of Cucurbitacin B in T2R10 (FIG. 8). Alternatively, orin addition, in certain embodiments, the amino acids that the compoundsinteract with comprise one or more of T2R10 residues Lys170, Glu172, andAsn181, either alone or in conjunction with interactions listed above,for example, by polar, hydrogen bonding, or charged interactions, asexemplified by in silico modeling of Cucurbitacin B in T2R10 (FIG. 8).Alternatively, or in addition, in certain embodiments, the amino acidsthat the compounds interact with comprise any one, two, three or more ofthe T2R10 residues Lys258, Leu180, Lys170, Glu172, Asn181, Phe261,Met265, Ile262, Gln169, Lys69, Met168, Ile245, Val90, Phe242, Gln94,Val184, Asn93, Trp89, and Tyr241, for example, by polar, hydrogen bond,salt bridge, van der Waals, pi, or other interactions, as exemplified byin silico modeling of Cucurbitacin B in T2R10 (FIG. 8).

In one embodiment, the bitter taste receptor is a canine T2R43. Incertain embodiments, the amino acids that the compounds interact withcomprise one or more of T2R43 residues Tyr241, Trp88, and Thr181, forexample, by hydrogen bonding interactions, as exemplified by in silicomodeling of Propylthiouracil in T2R43 (FIG. 9). Alternatively, or inaddition, in certain embodiments, the amino acids that the compoundsinteract with comprise one or more of T2R43 residues Met177, Asn92,Asn184, and Phe185, either alone or in conjunction with interactionslisted above, for example, by polar or hydrogen bonding interactions, asexemplified by in silico modeling of Propylthiouracil in T2R43 (FIG. 9).Alternatively, or in addition, in certain embodiments, the amino acidsthat the compounds interact with comprise any one, two, three or more ofthe T2R43 residues Tyr241, Trp88, Thr181, Met177, Asn92, Asn184, Phe185,Gln152, His143, Phe261, Ala172, His85, Asp170, Lys265, Phe242, Leu245,Thr89, and Phe180, for example, by polar, hydrogen bond, salt bridge,van der Waals, pi, or other interactions, as exemplified by in silicomodeling of Propylthiouracil in T2R43 (FIG. 9).

In certain embodiments, the compounds interact with any one or more ofthe canine T2R receptors described herein, wherein the compoundsinteract with one or more amino acid residues present in the 7TM domainsof said receptors. The EC2 loop of said receptors is at the entrance tothe active site pocket of the receptors. In certain embodiments, aminoacid residues present in the EC2 loop of the bitter receptors interactwith the compounds described herein.

3.3 In Vitro Methods

The presently disclosed subject matter further provides in vitro methodsfor identifying raw materials for generating pet food, food products, orcompounds that can modulate the activity and/or expression of a bittertaste receptor.

Bitter taste receptors for use in the presently disclosed methods caninclude isolated or recombinant bitter taste receptors or cellsexpressing a bitter taste receptor, disclosed herein. In certainembodiments, the bitter taste receptor for use in the disclosed methodscan comprise the amino acid sequence of any one of SEQ ID NO:17-32, or afragment or variant thereof. In certain embodiments, the bitter tastereceptor for use in the disclosed method can have at least about 33%,34%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 65%, 70%, 72%, 75%, 79%, 80%,84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of any one of SEQ ID NO: 17-32, or afragment or variant thereof. In certain embodiments, the bitter tastereceptor for use in the disclosed method can be encoded by a nucleotidesequence comprising any one of SEQ ID NO: 1-16, or a fragment or variantthereof. In certain embodiments, the bitter taste receptor for use inthe presently disclosed subject matter can include a receptor encoded bya nucleotide sequence having at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to any one of SEQ IDNO: 1-16, or a fragment or variant thereof.

In certain embodiments, the method for identifying compounds thatmodulate the activity and/or expression of a bitter taste receptorcomprises measuring the biological activity of a bitter taste receptorin the absence and/or presence of a test compound. In certainembodiments, the method can include measuring the biological activity ofa bitter taste receptor in the presence of varying concentrations of thetest compound. The method can further include identifying the testcompounds that result in a modulation of the activity and/or expressionof the bitter taste receptor compared to the activity and/or expressionof the bitter taste receptor in the absence of the test compound.

In certain embodiments, the method can further include analyzing two ormore, three or more or four or more test compounds in combination. Incertain embodiments, the two or more, three or more or four or more testcompounds can be from different classes of compounds, e.g., amino acidsand small chemical compounds. For example, and not by way of limitation,the method can include analyzing the effect of one or more smallchemical test compounds on the biological activity and/or expression ofa bitter taste receptor in the presence of one or more amino acid testcompounds. In certain embodiments, the method for identifying the effectof a compound on the activity and/or expression of a bitter tastereceptor comprises analyzing the effect of a test compound on thebiological activity and/or expression of a bitter taste receptor in thepresence of a bitter taste receptor ligand, for example, a bittertastant or bitter receptor agonist.

In certain embodiments, the method for identifying compounds that canmodulate the activity and/or expression of a bitter taste receptorcomprises expressing a bitter taste receptor in a cell line andmeasuring the biological activity of the receptor in the presence and/orabsence of a test compound. The method can further comprise identifyingtest compounds that modulate the activity of the receptor by determiningif there is a difference in receptor activation in the presence of atest compound compared to the activity of the receptor in the absence ofthe test compound. In certain embodiments, the method can includemeasuring the biological activity of the bitter taste receptor in thepresence of varying concentrations of the test compound. In certainembodiments, the selectivity of the putative bitter taste receptormodulator can be evaluated by comparing its effects on other GPCRs ortaste receptors, e.g., umami, fatty acid, kokumi (CaSR), T1R, etc.receptors.

In certain embodiments, the compounds identified according to themethods described herein increase or decrease the biological activity ofa bitter taste receptor by at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more, compared to the biological activity of the bitter tastereceptor when the compound is not present.

In certain embodiments, the method for identifying compounds thatmodulate the activity and/or expression of a bitter taste receptorcomprises determining whether a compound modulates the receptordirectly, for example, as an agonist or antagonist. In certainembodiments, the method comprises determining whether a compoundindirectly modulates the activity of the receptor (e.g., as anallosteric modulator), for example, by enhancing or decreasing theeffect of other compounds on activating or inhibiting receptor activity.

Activation of the receptor in the presently disclosed methods can bedetected through the use of a labelling compound and/or agent. Incertain embodiments, the activity of the bitter taste receptor can bedetermined by the detection of secondary messengers such as, but notlimited to, cAMP, cGMP, IP3, DAG or calcium. In certain embodiments, theactivity of the bitter taste receptor can be determined by the detectionof the intracellular calcium levels. Monitoring can be by way of, butnot limited to, luminescence or fluorescence detection, such as by acalcium sensitive fluorescent dye or luminescent photoprotein. Incertain embodiments, monitoring can be by way of luminescence. Incertain embodiments, the intracellular calcium levels can be determinedusing a cellular dye, e.g., a fluorescent calcium indicator such asCalcium 4. In certain embodiments, the intracellular calcium levels canbe determined by measuring the level of calcium binding to acalcium-binding protein, for example, calmodulin. Alternatively and/oradditionally, the activity of the bitter taste receptor can bedetermined by the detection of the phosphorylation, transcript levelsand/or protein levels of one or more downstream protein targets of thebitter taste receptor.

The cell line used in the presently disclosed methods can include anycell type that is capable of expressing a bitter taste receptor (e.g.,stable or transient expression). Non-limiting examples of cells that canbe used in the disclosed methods include HeLa cells, Chinese hamsterovary cells (CHO cells), African green monkey kidney cells (COS cells),Xenopus oocytes, HEK-293 cells and murine 3T3 fibroblasts. In certainembodiments, the method can include expressing a bitter taste receptorin HEK-293 cells. In certain embodiments, the method can includeexpressing a bitter taste receptor in COS cells. In certain embodiments,the cells constitutively express the bitter taste receptor. In certainembodiments, the cells transiently express the bitter taste receptor. Inanother embodiment, expression of the bitter taste receptor by the cellsis inducible.

In certain embodiments, the cell expresses a calcium-bindingphotoprotein, wherein the photoprotein luminesces upon binding calcium.In certain embodiments, the calcium binding photoprotein comprises theprotein clytin. In certain embodiments the clytin is a recombinantclytin. In certain embodiments, the clytin comprises an isolated clytin,for example, a clytin isolated from Clytia gregarium. In certainembodiments, the calcium-binding photoprotein comprises the proteinaequorin, for example, a recombinant aequorin or an isolated aequorin,such as an aequorin isolated from Aequorea victoria. In certainembodiments, the calcium-binding photoprotein comprises the proteinobelin, for example, a recombinant obelin or an isolated obelin, such asan obelin isolated from Obelia longissima.

In certain embodiments, expression of a bitter taste receptor in a cellcan be performed by introducing a nucleic acid encoding a bitter tastereceptor into the cell. For example, and not by way of limitation, anucleic acid having the nucleotide sequence set forth in any one of SEQID NO: 1-16, or a fragment thereof, can be introduced into a cell. Incertain embodiments, the introduction of a nucleic acid into a cell canbe carried out by any method known in the art, including but not limitedto transfection, electroporation, microinjection, infection with a viralor bacteriophage vector containing the nucleic acid sequences, cellfusion, chromosome-mediated gene transfer, microcell-mediated genetransfer, spheroplast fusion, etc. Numerous techniques are known in theart for the introduction of foreign genes into cells (see, e.g.,Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al.,Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92(1985), the disclosures of which are hereby incorporated by reference intheir entireties) and can be used in accordance with the disclosedsubject matter. In certain embodiments, the technique can provide forthe stable transfer of the nucleic acid to the cell, so that the nucleicacid is expressible by the cell and inheritable and expressible by itsprogeny.

In certain embodiments, the technique can provide for a transienttransfer of the nucleic acid to the cell, so that the nucleic acid isexpressible by the cell, wherein the concentration of the nucleic acidand the expression decrease in subsequent generations of the cell'sprogeny.

In certain embodiments, the methods can include identifying compoundsthat bind to a bitter taste receptor. The methods can comprisecontacting a bitter taste receptor with a test compound and measuringbinding between the compound and the bitter taste receptor. For example,and not by way of limitation, the methods can include providing anisolated or purified bitter taste receptor in a cell-free system, andcontacting the receptor with a test compound in the cell-free system todetermine if the test compound binds to the bitter taste receptor. Incertain embodiments, the method can comprise contacting a bitter tastereceptor expressed on the surface of a cell with a candidate compoundand detecting binding of the candidate compound to the bitter tastereceptor. The binding can be measured directly, e.g., by using a labeledtest compound, or can be measured indirectly. In certain embodiments,the detection comprises detecting a physiological event in the cellcaused by the binding of the compound to the bitter taste receptor,e.g., an increase in the intracellular calcium levels. For example, andnot by way of limitation, detection can be performed by way offluorescence detection, such as a calcium sensitive fluorescent dye, bydetection of luminescence, or any other method of detection known in theart.

In other non-limiting embodiments, the in vitro assay comprises cellsexpressing a bitter receptor that is native to the cells. Examples ofsuch cells expressing a native bitter receptor include, for example butnot limited to, dog and/or cat taste cells (e.g., primary taste receptorcells). In certain embodiments, the dog and/or cat taste cellsexpressing a bitter receptor are isolated from a dog and/or cat andcultured in vitro. In certain embodiments, the taste receptor cells canbe immortalized, for example, such that the cells isolated from a dogand/or cat can be propagated in culture.

In certain embodiments, expression of a bitter taste receptor in a cellcan be induced through gene editing, for example, through use of theCRISPR gene editing system to incorporate a bitter taste receptor geneinto the genome of a cell, or to edit or modify a bitter taste receptorgene native to the cell.

In certain embodiments, the in vitro methods of identifying a compoundthat binds to a T2R comprises determining whether a test compoundinteracts with one or more amino acids of a T2R binding pocket, asdescribed herein.

In certain embodiments, compounds identified as modulators of a bittertaste receptor can be further tested in other analytical methodsincluding, but not limited to, in vivo assays, to confirm or quantitatetheir modulating activity.

In certain embodiments, the methods of identifying a bitter tastereceptor modulator can comprise comparing the effect of a test compoundto a bitter taste receptor agonist or antagonist. For example, a testcompound that increases or decreases the activity of the receptor in thepresence of an agonist when compared to the activity of the receptorwhen contacted with a bitter taste receptor agonist alone can beselected as a bitter taste receptor modulating compound.

Bitter receptor agonists that can be used according to said methods cancomprise one or more compounds described by Table 1.

TABLE 1 Canine Bitter Taste Receptor Agonists Compound: Chemicalstructure: Menthol

Ofloxacin

Chloroquine

Colchicine

1,10-phenanthroline

Cucurbitacin B

Propylthiouracil

In certain embodiments, the bitter taste receptor modulators of thepresent disclosure comprise a salt of the bitter taste receptormodulator, for example, but not limited to, an acetate salt or a formatesalt. In certain embodiments, the bitter taste receptor modulator saltcomprises an anion (−) (for example, but not limited to, Cl⁻, O²⁻, CO₃²⁻, HCO³⁻, OH⁻, NO³⁻, PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, HCOO⁻ and C₂O₄ ²⁻) bondedvia an ionic bond with a cation (+) (for example, but not limited to,Al³⁺, Ca²⁺, Na⁺, K⁺, Cu²⁺, H⁺, Fe³⁺, Mg²⁺, NH₄ ⁺ and H₃O⁺). In otherembodiments, the bitter taste receptor agonist salt comprises a cation(+) bonded via an ionic bond with an anion (−).

In certain embodiments, the bitter taste receptor modulators of thepresent application are identified through in silico modeling of abitter taste receptor, e.g., a canine bitter taste receptor, wherein thebitter taste receptor modulators of the present application comprise astructure that fits within a binding site of the bitter taste receptor.In certain embodiments, the in silico method comprises the in silicomethods described above and in the Examples section of the presentapplication.

In certain embodiments, the bitter taste receptor modulators of thepresent application are identified through an in vitro method, whereinthe bitter taste receptor modulator compounds modulate a bitter tastereceptor, disclosed herein, expressed by cells in vitro. In certainembodiments, the in vitro method comprises the in vitro methodsdescribed above and in the Examples section of the present application.

4. Pet Food Products

The present application provides for screening methods that can be usedto identify suitable raw materials to produce a palatable and nutritiouspet food product. The presently disclosed screening methods can also beused to determine if a finished pet food product would be palatable tothe pet (e.g., a dog). For example, the in vitro methods describedherein can be used to screen raw materials and finished pet foodproducts to identify whether the raw materials or finished pet foodproducts comprise compounds that modulate bitter receptor activityand/or expression. In certain embodiments, raw materials and finishedpet food products that do not increase the activity and/or expression ofa bitter receptor can be selected for use in, or as, a pet food productfor consumption. Non-limiting examples of suitable pet food productsinclude wet food products, dry food products, moist food products, petfood supplements (e.g., vitamins), pet beverage products, snack andtreats and pet food categories described herein.

One of the goals of the pet care industry is to identify sustainableprotein sources for pets that do not compete with the human food chain.As such, there is an ongoing search for novel protein sources that fitthese criteria. The presently disclosed screening method can be used toidentify which of the novel protein sources would be consideredpalatable to the pet, or at least have no effect on the palatability ofthe other ingredients of the pet food. In certain embodiments, the novelprotein source (i.e., raw material) is meat, fish, cheese, beans, yeast,yeast extracts, bacteria, algae, fungi, nuts, seeds or other plantmaterial, or combinations thereof. In certain embodiments, the rawmaterial is meat.

In certain embodiments, the protein source can be derived from a varietyof plant sources. Non-limiting examples of plant sources include corn,maize, rice, soy, wheat, etc. For example, and not by way of limitation,the plant-derived protein can include lupin protein, wheat protein, soyprotein and combinations thereof. Alternatively or additionally, theprotein source can be derived from a variety of animal sources, forexample, a multicellular eukaryotic organism from the kingdom animalia.Non-limiting examples of animal protein include beef, pork, poultry,lamb or fish including, for example, muscle meat, meat byproduct, meatmeal or fish meal. Other non-limiting examples of animal sources includeinsects, or other organism from the phylum arthropoda.

In certain embodiments, the protein source can be derived from yeast orany other single-cell eukaryotic organisms, mold, mushroom or fungi.

In certain embodiments, the protein source can be derived from bacteria,archaea, or any other archaebacteria, eubacteria, or prokaryoticorganism.

In certain embodiments, the protein source can be derived from algae,kelp, seaweed, or any other single or multicellular photosyntheticorganism or protist.

In certain embodiments, the presently disclosed subject matter includesaccepting or rejecting a raw material for the production of pet foodbased on the raw material's ability to enhance, increase, decreaseand/or modulate the activity and/or expression of a bitter tastereceptor. In certain embodiments, the raw material is rejected if theraw material results in the enhancement or increase in the activityand/or expression of at least one bitter taste receptor. In certainembodiments, the raw material is rejected if the raw material results inthe enhancement or increase in the activity and/or expression of atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least ten, at leasteleven, at least twelve, at least thirteen, at least fourteen, at leastfifteen, and/or at least sixteen bitter taste receptors. In certainembodiments, the raw material is accepted if it does not modulate theactivity of at least one bitter taste receptor. In certain embodiments,the raw material is selected if it inhibits or blocks the activityand/or expression of at least one bitter taste receptor. In certainembodiments, the bitter receptor is selected from any one or more ofcanine T2R1, T2R2, T2R3, T2R4, T2R5, T2R7, T2R10, T2R12, T2R38, T2R39,T2R40, T2R41, T2R42, T2R43, T2R62, and/or T2R67.

In certain non-limiting embodiments, a raw material that results in theenhancement or increase in the activity and/or expression of at leastone bitter taste receptor can be admixed with a compound that inhibitsor reduces the activity and/or expression of the at least one bitterreceptor, wherein the admixture is accepted for the production of petfood.

During the production of pet food, some of the materials may change formdue to mechanical forces, thermal forces, or chemical reactions. Thepresently disclosed screening method can be used to identify pet foodproducts that form compounds that are unpalatable to an animal, forexample, a canine, for example, a compound that enhances or increasesthe activity and/or expression of a bitter receptor.

In certain embodiments, the presently disclosed subject matter includesaccepting or rejecting a pet food product based on the product's abilityto enhance, increase, decrease and/or modulate the activity and/orexpression of a bitter taste receptor. In certain embodiments, the petfood product is rejected if the product results in the enhancement orincrease in the activity and/or expression of at least one bitter tastereceptor. In certain embodiments, the pet food product is rejected ifthe product results in the enhancement or increase in the activityand/or expression of at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,at least ten, at least eleven, at least twelve, at least thirteen, atleast fourteen, at least fifteen, and/or at least sixteen bitter tastereceptors. In certain embodiments, the pet food product is accepted ifit does not modulate the activity of at least one bitter taste receptor.In certain embodiments, the pet food product is selected if it inhibitsor blocks the activity and/or expression of at least one bitter tastereceptor. In certain embodiments, the bitter receptor is selected fromany one or more of canine T2R1, T2R2, T2R3, T2R4, T2R5, T2R7, T2R10,T2R12, T2R38, T2R39, T2R40, T2R41, T2R42, T2R43, T2R62, and/or T2R67.

The flavor compositions of the present disclosed subject matter can alsobe used in a wide variety of pet food products. The combination of theflavoring composition(s) of the presently disclosed subject mattertogether with a pet food product and optional ingredients, when desired,provides a flavoring agent that possesses unexpected taste and imparts,for example, a desirable bitter sensory experience. The flavorcompositions disclosed herein can be added prior to, during or afterformulation processing or packaging of the pet food product, and thecomponents of the flavor composition can be added sequentially orsimultaneously.

In certain embodiments, the pet food product is a nutritionally completedry, wet or semi-moist food product. A dry or low moisture-containingnutritionally-complete pet food product can comprise less than about 15%moisture. A wet or high moisture-containing nutritionally-complete petfood product can comprise greater than about 50% moisture. Such foodproducts can include from about 10% to about 90% fat, from about 10% toabout 70% protein and from about 5% to about 80% carbohydrates, e.g.,dietary fiber and ash, on a percent energy basis.

In certain embodiments, the pet food product is a nutritionally completedry, wet or semi-moist food product. In certain embodiments, the petfood product includes from about 60% fat, from about 30% protein andfrom about 10% carbohydrates, e.g., dietary fiber and ash, on a percentenergy basis.

In certain embodiments, the pet food product is a nutritionally completemoist food product. A moist, e.g., semi-moist or semi-dry or soft dry orsoft moist or intermediate or medium moisture containingnutritionally-complete pet food product comprises from about 15 to about50% moisture.

In certain embodiments, the pet food product is a pet food snackproduct. Non-limiting examples of pet food snack products include snackbars, pet chews, crunchy treats, cereal bars, snacks, biscuits and sweetproducts.

In certain embodiments of the present disclosure, the taste and/orpalatability attributes of a pet food product or medicine preparedaccording to the methods described herein can be measured by an in vivotasting method that uses a panelist of taste testers. For example, butnot by way of limitation, the panel can contain canine panelists. Incertain embodiments, the palatability of a pet food product containing,for example, a screened raw material or a screened pet food product canbe determined by the consumption of the pet food product alone (e.g.,the one bowl test, monadic ranking). In certain embodiments, thepalatability of a screened raw material or a screened pet food productcan be determined by the preferential consumption of the pet foodproduct or raw material, versus a pet food product that is known to bepalatable to the animal (e.g., the two bowl test for testing preference,difference and/or choice).

In certain embodiments, the palatability and/or bitter blocking taste ofa compound identified according to the methods described herein can bedetermined by the preferential consumption of a water solutioncontaining said compound versus a water solution that does not containthe compound or contains a different flavor composition, for example, abitter receptor agonist (e.g., the two bottle test). The intake ratiofor each pet food product or water solution can be determined bymeasuring the amount of one ration consumed divided by the totalconsumption. The consumption ratio (CR) can then be calculated tocompare the consumption of one ration in terms of the other ration todetermine the preferential consumption of one food product or watersolution over the other. Alternatively or additionally, the differencein intake (g) can be used to assess the average difference in intakebetween the two solutions in a two bottle test or between two pet foodproducts in a two bowl test at a selected significance level, forexample, at the 5% significance level to determine an average differencein intake with a 95% confidence interval. In certain embodiments, theconfidence interval can be about 90%. However, any significance levelmay be used, for example, a 1, 2, 3, 4, 5, 10, 15, 20, 25 or 50%significance level.

In certain embodiments, percentage preference scores, e.g., thepercentage preference for one solution or food product by an animal, isthe percentage of the total liquid or food product ingested during thetest that that solution or food product accounts for, can also becalculated.

5. Taste Deterrents

The present disclosure provides methods for maintaining the health of ananimal by imparting a bitter taste and/or decreasing the palatability ofan object or surface. In certain embodiments, the method comprisesapplying, coating or contacting a taste deterrent product comprising acompound identified according to the methods described herein to theobject or surface, and thereby preventing ingestion of said object orsurface by an animal. Accordingly, detrimental effects on the animal'shealth that could result from ingestion of said object or surface areavoided. In certain embodiments, the object or surface is harmful to thehealth of the animal or toxic to the animal.

EXAMPLES

The presently disclosed subject matter will be better understood byreference to the following Examples, which are provided as exemplary ofthe invention, and not by way of limitation.

Example 1 In Silico Model of Interactions Between Canine T2R Receptorsand Putative Binding Compounds

The present example describes the computational modeling of caninebitter taste receptors (T2Rs) to identify putative bitter taste receptormodulators.

Homology models of canine T2R receptors were based on crystal structureof 3SN6 from Protein Data Bank (PDB). 3SN6 is the crystal structure ofβ2 adrenergic receptor from Group A GPCR with bound agonist (BI-167107from Boehringer Ingelheim). (Rasmussen et al., Nature, 477: 549-555(2011)). The models were built using the I-TASSER Suite of programs(Yang et al., Nat Methods, 12: 7-8 (2015)) and Modeller (Eswar et al.,Curr Protoc Bioinformatics, 15: 5.6.1-5.6.30 (2006)), which is part ofthe DiscoveryStudio (DS) suite of programs from Accelrys(DiscoveryStudio (DS) is suite of interactive modeling and simulationprograms from the Accelrys corporation).

The bitter compounds were docked into the active site of canine bitterreceptors. The docking program BioDock from BioPredict, Inc., was usedbut other state of the art docking programs could be used for thispurpose.

The results of in silico modeling are presented in FIGS. 3-9.

Example 2 Identification of Canine Bitter Receptor (T2R) ModulatorsUsing in Vitro Assays.

The present example describes an in vitro assay for identifyingcompounds that modulate the activation of the canine bitter tastereceptor (T2R).

Compounds identified by in silico modeling with a bitter taste receptor,as detailed above in Example 1, as putative bitter taste receptormodulators will be selected for further testing in vitro. In vitrofunctional characterization of the selected modulators will be used toevaluate the effectiveness of a putative modulator compound inactivating or inhibiting the bitter taste receptor.

HEK293 cells (or other suitable expression system) that stably ortransiently express a canine bitter taste receptor (e.g., canine T2R1,T2R2, T2R3, T2R4, T2R5, T2R7, T2R10, T2R12, T2R38, T2R39, T2R40, T2R41,T2R42, T2R43, T2R62, or T2R67) will be exposed to putative compounds tomodulate the activity and/or expression of the bitter taste receptor.

An exemplary method of an in vitro assay is as follows. All transienttransfections will be performed with, for example, Lipofectamine2000(Invitrogen) according to the manufactures protocol. 10 μlLipofectamine2000 will be diluted in 500 μl DMEM (Life Technologies) andincubated for 5 minutes at room temperature. 3 μg of plasmid DNA (1μg/μl) will be diluted in 500 μl DMEM and added to the Lipofectamine2000mix to obtain a final volume of 1000 μl. After additional 30 minutes ofincubation at room temperature, the DNA-Lipofectamine complex will beadded to 1000 μl of a cell suspension containing 1,400,000 cells/ml.Subsequently, 25 μl of the complete mixture will be seeded into eachwell of a black 384 well polystyrene assay plate. At 3 hourspost-transfection the transfection mix will be removed from the cellsand fresh DMEM containing 10% FBS and 1% P/S will be added. At 27 to 30hours post-transfection the medium will be removed from the cells and 20μl loading buffer that includes a calcium sensitive fluorescent orluminescent dye (Tyrode's buffer+2 μM Fluo4-AM (Invitrogen)+2.5 mMprobenecid (Invitrogen) for fluorescence or Coelenterazine(Biosynth)+Tyrode's buffer for luminescence) will be added for 1 hour(fluorescence) or 3 hours (luminescence) at 37° C. The cells will thenbe washed 2 times every 20 minutes with Tyrode's buffer using anautomated plate washer (Biochrom Asys Plate Washer) for the fluorescentprotocol. No wash step will be required for the luminescent protocol.

Activation of the bitter taste receptor will then be detected, forexample, by detecting a change in intracellular calcium levels using thecalcium sensitive fluorescent dye, the calcium sensitive luminescentphotoprotein, or by any detection system known in the art. Cells that donot express the bitter taste receptor (MOCK control) will be used as acontrol. Examples of such data capturing systems include FLIPR® Tetra ora FlexStation® 3 system. However, other imaging techniques and systemscan be used, for example, microscopic imaging of the treated cells.

For each putative bitter taste receptor modulator, dose response curveswill be generated with at least 8 concentrations in triplicate and theEC₅₀ so value of the putative bitter taste receptor modulator will bedetermined. Graphs will be plotted, for example, in SigmaPlot V12(Systat Software) with error bars representing standard error. The termhalf maximal effective concentration (EC₅₀) refers to the concentrationof a compound which induces a response halfway between the baseline andthe maximum after a specified exposure time.

Example 3 Identification of Canine Bitter Receptor (T2R) ModulatorsUsing in Vitro Assays.

The present example describes an in vitro assay for identifyingcompounds that modulate the activation of the canine bitter tastereceptor (T2R) by a T2R ligand.

Compounds identified by in silico modeling with a bitter taste receptor,as detailed above in Example 1, as putative bitter taste receptormodulators will be selected for further testing in vitro. In vitrofunctional characterization of the selected modulators will be used toevaluate the effectiveness of a putative modulator compound inmodulating the activation of the bitter taste receptor by a bitter tastereceptor ligand.

HEK293 cells (or other suitable expression system) that stably ortransiently express a canine bitter taste receptor (e.g., canine T2R1,T2R2, T2R3, T2R4, T2R5, T2R7, T2R10, T2R12, T2R38, T2R39, T2R40, T2R41,T2R42, T2R43, T2R62, or T2R67) will be exposed to putative modulatorcompounds and a bitter taste receptor ligand (e.g., an agonist) tomodulate the activity and/or expression of the bitter taste receptor.

An exemplary method of an in vitro assay is as follows. All transienttransfections will be performed with, for example, Lipofectamine2000(Invitrogen) according to the manufactures protocol. 10 μlLipofectamine2000 will be diluted in 500 μl DMEM (Life Technologies) andincubated for 5 minutes at room temperature. 3 μg of plasmid DNA (1μg/μl) will be diluted in 500 μl DMEM and added to the Lipofectamine2000mix to obtain a final volume of 1000 μl. After additional 30 minutes ofincubation at room temperature, the DNA-Lipofectamine complex will beadded to 1000 μl of a cell suspension containing 1,400,000 cells/ml.Subsequently, 25 μl of the complete mixture will be seeded into eachwell of a black 384 well polystyrene assay plate. At 3 hourspost-transfection the transfection mix will be removed from the cellsand fresh DMEM containing 10% FBS and 1% P/S will be added. At 27 to 30hours post-transfection the medium will be removed from the cells and 20μl loading buffer that includes a calcium sensitive fluorescent dye orluminescent substrate (Tyrode's buffer+2 μM Fluo4-AM (Invitrogen)+2.5 mMprobenecid (Invitrogen) for fluorescence or Coelenterazine (Biosynth)+Tyrode's buffer for luminescence) will be added for 1 hour(fluorescence) or 3 hours (luminescence) at 37° C. The cells will thenbe washed 2 times every 20 minutes with Tyrode's buffer using anautomated plate washer (Biochrom Asys Plate Washer) for the fluorescentprotocol. No wash step will be required for the luminescent protocol.

Activation of the bitter taste receptor will then be detected, forexample, by detecting a change in intracellular calcium levels using thecalcium sensitive fluorescent dye, the calcium sensitive luminescentphotoprotein, or by any detection system known in the art. Cells that donot express the bitter taste receptor (MOCK control) will be used as acontrol. Examples of such data capturing systems include FLIPR® Tetra ora FlexStation® 3 system. However, other imaging techniques and systemscan be used, for example, microscopic imaging of the treated cells.

For each putative bitter taste receptor modulator, dose response curveswill be generated with at least 8 concentrations in triplicate and theEC₅₀ value of the putative bitter taste receptor modulator will bedetermined. Graphs will be plotted, for example, in SigmaPlot V12(Systat Software) with error bars representing standard error.

Example 4 BLAST Search Homology Comparison of Canine T2R Receptors andHuman T2R Receptors.

A BLAST search was conducted to compare certain canine T2R amino acidsequences with human T2R amino acid sequences. BlastP was used withparameters set as follows: Matrix Blosum 62; gap existence cost 11; gapextension cost 1; and use of compositional score matrix adjustment.

T2R2 is shared by dog and cat, but not human. A BLAST search comparisonof the canine T2R2 amino acid sequence with human T2R amino acidsequences shows that the canine T2R2 was equidistant from every humanT2R bitter receptor tested (Table 2).

TABLE 2 BLAST Search Homology Comparison of Canine T2R2 to Human T2R MaxTotal Query E Sequence Description score score Cover value Identitytaste receptor type 2 member 7 172 172 93% 3e−50 35% [Homo sapiens]taste receptor type 2 member 9 158 158 97% 9e−45 33% [Homo sapiens]taste receptor type 2 member 10 143 143 97% 4e−39 32% [Homo sapiens]taste receptor type 2 member 8 141 141 97% 1e−38 33% [Homo sapiens]taste receptor type 2 member 41 134 134 93% 9e−36 31% [Homo sapiens]taste receptor type 2 member 13 133 133 97% 1e−35 29% [Homo sapiens]taste receptor type 2 member 1 129 129 95% 5e−34 32% [Homo sapiens]taste receptor type 2 member 42 129 129 97% 6e−34 34% [Homo sapiens]taste receptor type 2 member 39 120 120 96% 1e−30 29% [Homo sapiens]taste receptor type 2 member 5 117 117 97% 1e−29 31% [Homo sapiens]taste receptor type 2 member 60 115 115 91% 6e−29 30% [Homo sapiens]taste receptor type 2 member 43 115 115 93% 7e−29 32% [Homo sapiens]taste receptor type 2 member 30 115 115 96% 9e−29 32% [Homo sapiens]taste receptor type 2 member 45 113 113 96% 3e−28 31% [Homo sapiens]taste receptor type 2 member 40 112 112 96% 1e−27 30% [Homo sapiens]taste receptor type 2 member 3 111 111 95% 2e−27 32% [Homo sapiens]taste receptor type 2 member 16 109 109 95% 6e−27 28% [Homo sapiens]taste receptor type 2 member 31 107 107 92% 3e−26 33% [Homo sapiens]taste receptor type 2 member 46 104 104 96% 4e−25 30% [Homo sapiens]taste receptor type 2 member 19 102 102 94% 2e−24 28% [Homo sapiens]taste receptor type 2 member 38 102 102 95% 2e−24 28% [Homo sapiens]taste receptor type 2 member 50 100 100 76% 8e−24 31% [Homo sapiens]taste receptor type 2 member 20 100 100 96% 1e−23 29% [Homo sapiens]taste receptor type 2 member 4 99.8 99.8 94% 2e−23 29% [Homo sapiens]taste receptor type 2 member 14 99.4 99.4 94% 2e−23 28% [Homo sapiens]

T2R12 is shared by dog and cat, but not human. A BLAST search comparisonof the canine T2R12 amino acid sequence with human T2R amino acidsequences shows that the canine T2R12 was equidistant from every humanT2R bitter receptor tested (Table 3).

TABLE 3 BLAST Search Homology Comparison of Canine T2R12 to Human T2RMax Total Query E Sequence Description score score Cover value Identitytaste receptor type 2 member 7 196 196 98% 3e−59 40% [Homo sapiens]taste receptor type 2 member 8 188 188 96% 2e−56 41% [Homo sapiens]taste receptor type 2 member 9 168 168 100%  1e−48 39% [Homo sapiens]taste receptor type 2 member 10 160 160 99% 1e−45 38% [Homo sapiens]taste receptor type 2 member 30 157 157 95% 2e−44 39% [Homo sapiens]taste receptor type 2 member 46 150 150 97% 5e−42 36% [Homo sapiens]taste receptor type 2 member 13 150 150 99% 6e−42 35% [Homo sapiens]taste receptor type 2 member 14 150 150 99% 8e−42 38% [Homo sapiens]taste receptor type 2 member 43 149 149 94% 2e−41 38% [Homo sapiens]taste receptor type 2 member 45 147 147 97% 9e−41 34% [Homo sapiens]taste receptor type 2 member 3 136 136 100%  1e−36 37% [Homo sapiens]taste receptor type 2 member 31 133 133 94% 2e−35 36% [Homo sapiens]taste receptor type 2 member 20 130 130 97% 2e−34 35% [Homo sapiens]taste receptor type 2 member 19 127 127 97% 3e−33 36% [Homo sapiens]taste receptor type 2 member 42 126 126 98% 1e−32 35% [Homo sapiens]taste receptor type 2 member 50 122 122 94% 2e−31 35% [Homo sapiens]taste receptor type 2 member 4 95.5 95.5 94% 4e−22 30% [Homo sapiens]taste receptor type 2 member 1 95.1 95.1 93% 7e−22 31% [Homo sapiens]taste receptor type 2 member 41 89.7 89.7 91% 4e−20 32% [Homo sapiens]taste receptor type 2 member 5 86.3 86.3 96% 7e−19 27% [Homo sapiens]taste receptor type 2 member 39 85.9 85.9 95% 1e−18 29% [Homo sapiens]taste receptor type 2 member 38 85.5 85.5 98% 2e−18 30% [Homo sapiens]taste receptor type 2 member 40 78.2 78.2 96% 5e−16 30% [Homo sapiens]taste receptor type 2 member 60 72.4 72.4 91% 6e−14 26% [Homo sapiens]taste receptor type 2 member 16 57.0 57.0 81% 1e−08 27% [Homo sapiens]

Canine T2R62 is unique to dog when compared to humans and felines. Inparticular, canine T2R62 comprises an amino acid sequence that isdifferent from all human bitter receptors (Table 4).

TABLE 4 BLAST Search Homology Comparison of Canine T2R62 to Human T2RMax Total Query E Sequence Description score score Cover value Identitytaste receptor type 2 member 16 203 203 98% 1e−62 39% [Homo sapiens]taste receptor type 2 member 41 184 184 90% 7e−55 40% [Homo sapiens]taste receptor type 2 member 60 157 157 93% 1e−44 36% [Homo sapiens]taste receptor type 2 member 9 115 115 99% 3e−29 32% [Homo sapiens]taste receptor type 2 member 1 115 115 97% 3e−29 31% [Homo sapiens]taste receptor type 2 member 46 115 115 98% 5e−29 30% [Homo sapiens]taste receptor type 2 member 13 115 115 96% 5e−29 30% [Homo sapiens]taste receptor type 2 member 7 115 115 98% 5e−29 30% [Homo sapiens]

Example 5 Identification of Canine Bitter Receptor (T2R) ModulatorsUsing in Vitro Assays.

Compounds identified by in silico modeling with a bitter taste receptor,as detailed above in Example 1, were selected for further testing invitro. In vitro functional characterization of the selected modulatorswas used to evaluate the effectiveness of the putative modulatorcompounds in modulating the activation of the bitter taste receptors.

HEK293 cells that transiently expressed a canine bitter taste receptorselected from canine T2R1, T2R2, T2R3, T2R4, T2R5, T2R10, and T2R43,were exposed to compounds to determine whether the compounds modulatedthe activity of the bitter taste receptors.

All transient transfections were performed with Lipofectamine2000(Invitrogen) according to the manufactures protocol. 10 μlLipofectamine2000 were diluted in 500 μl DMEM (Life Technologies) andincubated for 5 minutes at room temperature. 3 μg of plasmid DNA (1μg/μl) was diluted in 500 μl DMEM and added to the Lipofectamine2000 mixto obtain a final volume of 1000 μl. After an additional 30 minutes ofincubation at room temperature, the DNA-Lipofectamine complex was addedto 1000 μl of a cell suspension containing 1,400,000 cells/ml.Subsequently, 25 μl of the complete mixture was seeded into each well ofa black 384 well polystyrene assay plate. At 3 hours post-transfectionthe transfection mix was removed from the cells and fresh DMEMcontaining 10% FBS and 1% P/S was added. At 27 to 30 hourspost-transfection the medium was removed from the cells and 20 μlloading buffer that included a calcium sensitive fluorescent dye orluminescent substrate (Tyrode's buffer+2 μM Fluo4-AM (Invitrogen)+2.5 mMprobenecid (Invitrogen) for fluorescence or Coelenterazine(Biosynth)+Tyrode's buffer for luminescence) were added for 1 hour(fluorescence) or 3 hours (luminescence) at 37° C. The cells were thenwashed 2 times every 20 minutes with Tyrode's buffer using an automatedplate washer (Biochrom Asys Plate Washer) for the fluorescent protocol.No wash step was required for the luminescent protocol.

Activation of the bitter taste receptor was determined by detecting achange in intracellular calcium levels as measured by fluorescence orluminescence of the calcium sensitive fluorescent dye or luminescentphotoprotein. Cells that did not express the bitter taste receptor (MOCKcontrol) were used as a control. A FLIPR® Tetra system was used tomeasure fluorescence or luminescence.

For each putative bitter taste receptor modulator, dose response curveswere generated with at least 8 concentrations in triplicate and the EC50value of the putative bitter taste receptor modulator was determined asshown in FIGS. 3-9. Graphs were plotted in SigmaPlot V12 (SystatSoftware) with error bars representing standard error.

Although the presently disclosed subject matter and its advantages havebeen described in detail, it should be understood that various changes,substitutions and alterations can be made herein without departing fromthe spirit and scope of the invention as defined by the appended claims.Moreover, the scope of the present application is not intended to belimited to the particular embodiments of the process, machine,manufacture, composition of matter, means, methods and steps describedin the specification. As one of ordinary skill in the art will readilyappreciate from the disclosure of the presently disclosed subjectmatter, processes, machines, manufacture, compositions of matter, means,methods, or steps, presently existing or later to be developed thatperform substantially the same function or achieve substantially thesame result as the corresponding embodiments described herein may beutilized according to the presently disclosed subject matter.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

Patents, patent applications, publications, product descriptions andprotocols are cited throughout this application the disclosures of whichare incorporated herein by reference in their entireties for allpurposes.

We claim:
 1. A method for modulating intensity of a bitter taste of apet food product, the method comprising: (a) providing the pet foodproduct; and (b) combining the pet food product with a compound inamount effective to modulate the bitter taste of the pet food product;wherein the compound binds to one or more amino acids of a canine bittertaste receptor T2R2 comprising an amino acid sequence set forth in SEQID NO: 18; and wherein the one or more amino acids of the canine bittertaste receptor T2R2 are selected from the group consisting of Ser94,Trp90, Lys268, Tyr245, Glu180, Arg176, Met91, Asn185, Val184, Met181,Phe249, Pro155, Gln177, Lys174, Phe264, Phe93, Leu59, Met271, Phe246,and Leu188.
 2. The method of claim 1, further comprising determiningbiological activity of the canine T2R2 bitter taste receptor.
 3. Themethod of claim 1, wherein the compound increases the intensity of abitter taste of a pet food product.
 4. The method of claim 1, whereinthe compound decreases the intensity of a bitter taste of a pet foodproduct.
 5. The method of claim 1, wherein the compound binds to one ormore amino acids selected from the group consisting of Ser94, Trp90,Lys268, Tur245, and Glu180.
 6. The method of claim 1, wherein thecompound binds to one or more amino acids selected from Art 176 or Met91.
 7. The method of claim 1, wherein the compound binds to one or moreamino acids selected from the group consisting of Asn185, Val184,Met181, Phe249, Pro155, Gln177, Lys174, Phe264, Phe93, Leu59, Met271,Phe246, and Leu188.
 8. The method of claim 1, wherein the compound bindsto two or more amino acids.
 9. The method of claim 1, wherein thecompound binds to three or more amino acids.
 10. The method of claim 1,wherein the compound binds to five or more amino acids.